PROJECT SUMMARY Our exciting new data show that tubulointerstitial fibrosis sensitizes glomeruli to any ensuing injury. We propose that this mechanism perpetuates CKD progression in human and experimental CKD. We further propose that this mechanism is critical regardless of whether the tubulointerstitial injury is the primary insult, or occurs secondarily in any disease setting. Our central hypothesis is that even mild, functionally recovered tubular injury sensitizes glomeruli to subsequent injury by decreasing podocyte repair mechanisms. This is critical because progressive glomerulosclerosis is tightly linked to loss of podocytes, which cannot replicate and replace themselves after severe injury. Instead, podocytes rely on replacement by progenitor cells including parietal epithelial cells, adjacent proximal tubules and cells of renin lineage present in the juxtaglomerular area. A current limitation to investigate mechanisms of tubular-glomerular crosstalk is that existing models injure either tubules or glomeruli or have simultaneous injury of both. We have developed a novel model to sequentially first injure tubules, with functional recovery, and then induce specific glomerular injury. Our tubular-glomerular injury model reveals that PECs near the urinary pole show more squamous and less columnar appearance after tubular injury, with increased plasminogen activator inhibitor-1 (PAI-1), suggesting progenitor PECs are affected by tubular injury. We have generated floxed PAI-1 mice, and inducible cell-specific PAI-1 knockdown mice as key tools to determine the cell-specific mechanisms of PAI- 1 actions in this podocyte repair/replenishment process. Our data suggest that podocyte- or PEC-PAI-1 knockdown promotes podocyte maintenance/repair in vivo, and that PAI-1-/- podocytes are resistant to injury in vitro. We also found that tubular injury increased macula densa nNOS, with more renin+ cell migration to the glomerular tuft after sequential tubular-glomerular injury. We will first determine the threshold for tubular injury to initiate an adverse cascade of progressive glomerular injury. We will next test the hypothesis that tubular injury decreases podocyte repair and determine the contribution of potential PEC progenitors in their repair progenitor capacities, using PEC lineage mice. We will add studies of the role of PAI-1 in PEC migration and differentiation to a podocyte-like or prosclerotic cell with cell-specific inducible knockdown of PAI- 1, and complementary in vitro studies. We will also assess the contribution of renin lineage cells to podocyte repair/replacement, and impact of tubular injury, and role of Notch in these potential progenitors. We will use renin lineage mice to directly assess the contribution of these potential progenitor cells to podocyte replacement after injury, using novel dual tagged mice, allowing tagging of renin lineage and of renin expression, with ex vivo analysis of the differentiation state of glomerular renin lineage cells. Together, our studies will determine effects of tubular injury on subsequent glomerular injury, and impact and mechanisms of this tubular injury on potential podocyte progenitor cells, using novel models and state of the art approaches.